The overall objective of this project is to determine the feasibility of using a combination immunization strategy to elicit protective immunity against primary isolates of HIV-1. To-date, only live attenuated virus vaccines have been shown to protect against highly pathogenic isolates of SIV propagated in primary macaque cells. However, because of the safety concerns, there remains a real and justifiable need for alternative approaches of immunization against HIV. In the current funding period, we showed that protective immunity could be achieved against both blood borne and mucosal infection by a pathogenic isolate of SIVmne by immunization with live recombinant virus priming followed by subunit antigen boosts. This combination immunization strategy was also effective against a chimeric simian-human immunodeficiency virus (SHIV) with envelope from a laboratory-adapted virus HIV-1IIIB. Based on these findings, we propose to extend our studies in the SHIV model and to determine if a similar strategy will be effective against immunodeficiency viruses with envelope from primary HIV-1 isolates. Our overall hypotheses are: (1) protection against primary isolates of HIV-1 can be induced by attenuated virus infection; (2) such protection is mediated by immune responses and is not dependent on sustained viral replication; and (3) protective immunity can be achieved by a combination immunization strategy with recombinant vaccines similar to those used in our previous work. To test these hypotheses, we propose in Specific Aim 1 to determine if limited viral replication resulted from anti-viral drug treatment during a live attenuated virus infection could effectively prime protective immune responses against a pathogenic SHIV with HIV-1 envelope. In Specific Aim 2, we will determine if such protective immunity could also be elicited by a combination immunization regimen with live vectored vaccine priming followed by recombinant antigen boosting. In Specific Aim 3, we will determine if the success or failure of the combination immunization regimen depends on the biological phenotype or the genotypic relatedness of the challenge viruses. Finally, in Specific Aim 4, we will examine if the potential limitations of the combination immunization approach could be overcome by enhancing the presentation of specific antigens by the recombinant vaccines. Results from these studies will provide further insight into the protective mechanisms against HIV-1 as well as vaccine strategies and prototype vaccine candidates for future clinical developments.